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Properties of Allosteric Enzymes
(1)
An allosteric enzyme possesses at least 2 spatially distinct binding
sites on the protein molecules the active or the catalytic site and the
regulator or the allosteric site. The metabolic regulator molecule
binds at the allosteric site and produces a change in the
conformational structure of the enzyme, so that the geometrical
relationship of the amino acid residues in the catalytic site is modified.
Consequently, the enzyme activity either increases (activation) or
decreases (inhibition).
(2) Allosteric enzymes show 2 different types of control – heterotropic
and homotropic depending on the nature of the modulating molecule.
Heterotropci enzymes are stimulated or inhibited by an effector or
modulator molecule other than their substrates, e.g. threonine
deaminase the substrate is threamine and the modulator is iso-leucine.
When the modulator promotes the binding of substrate to the allosteric
enzyme, the modulator is said to be a +ve effector or allosteric
activator whereas when the modulator diminishes the binding of
substrate, it is called a –ve effector or allosteric inhibitor, +ve
effectors increase the number of binding sites for substrate whereas –
ve effectors decrease the number of binding sites for the substrate.
In homotropic enzymes, the substrate also functions as the modulator.
Homotropic enzymes contain 2 or more binding sites for the substrate
modulation depends on how many of the substrate sites are bound.
(3) Their Kinetics do not obey the Michaelis – Mention equation. A plot
of V vs S for an allosteric enzyme yields sigmoid – or S-shaped curve
rather than rectangular hyperbola.
V-max
Hyper-bold
V
1
Sigmoid
S
One possible explanation for the occurrence of these sigmoid Kinetics is
that each molecule of enzyme possesses more than one catalytic site to
which the substrate could be bound. The binding of the regulator molecule
causes a conformational change in the protein so that the structure of the
catalytic site is modified. This conformational change can be considered as
information transfer between the regulator and the catalytic sites such that
the binding of substrate at one site affects the binding of subsequent
molecules of substrate at the remaining binding sites – a phenomenon
referred to as Cooperativity. When the binding of the regulator results in
more substrates being bound, then it is +ve cooperativity and the reverse as
–ve cooperativity.
(4) Inhibition of a regulatory enzyme does not conform to any normal
inhibition pattern and the inhibitor does not bear any obvious
structural relationship to the substrate. The enzyme exhibits extreme
specificity with regard to the regulator molecule.
(5) Allosteric enzymes have an oligomeric organization. They are
composed of more than one polypeptide chain and have more than
one S-binding site per enzyme molecule.
(6) Treatment of the allosteric enzyme with agents or conditions that exert
a mild denaturing effect can result in loss of sensitivity to the effects
of the regulatory molecule without changing the catalytic activity.
This phenomenon is referred to as desensitization and this can be
effected by high or low pH mercurials (such as mercuric chloride)
urea or by gentle heating. Desensitisation causes dissociation of the
ntive enzyme into its component sub-units and this prevents
interaction between the regulator and catalytic sites.
Unlike most enzymes, many allosteric enzymes undergo reversible
inactivation at OoC.
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(a) Covalently modulated enzymes.
This is a 2nd group of regulatory enzymes that are inter-converted
between active and inactive forms by other enzymes by covalent
modification of specific amino acid residues on the enzyme surface.
Covalent modification may either reinforce or counteract the effects of
allosteric regulators and hence may either intensify or tend to nullify
allosteric regulatory effects. Regulation by covalent modulation is well
documented in animals.
In mammalian systems, the 2 most common forms of covalent
modification are Partial proteolysis and Phosphorylation and Dephosphorylation. B/C Cells lack the ability to reunite the 2 portions of a
protein produced by hydrolysis of a peptide bond, proteolysis constitutes
an irreversible modification. In contrast, phosphorylation is a reversible.
Phosphorylation takes place on seryl, threomyl, or tyrosyl residues and it
is catalysed by a group of enzymes known as protein Kinases. B/c PO4
lation is versible, the hydrolytic removal of these phosphoryl groups is
also possible and it is catalysed by enzymes called protein phosphatases.
ATP
ADP
Mg
2+
Kinase
Phosphorylation
Enz – Ser – O – PO32-
Enz-Ser-OH
Phosphatase
Mg2+
3
Pi
Dephosphorylation H20
The activities of protein Kinases and protein Phosphatases are themselves
regulated; if not, their concerted action would be both thermodynamically
and biologically unproductive.
A classical example of an enzyme regulated by covalent modification of its
activity is glycogen phosphorylase of animal tissues which catalyses the
breakdown of glyzogen.
(Glucose)n
Glycogen
+
Pi
(Glucose)n-1
Shortened
glucogen
molecule
+
Glucose -1-
P04
Glycogen phosphorylase occurs in 2 forms, phosphorylase a, the more active
form and phosphory lase b, the less active form. Phosphorylase a is an
oligomeric protein with 4 major sub-units. Each sub-unit:
PO32
Ser
Ser-O-P032
Phosphorylase a (active)
Ser-O-P032
0
PO32
4
Phosphorylase
Phosphatase
4H20
4-ADP
4Pi
Phosphorylase
Kinase
4ATP
+
Phosphorylase b (inactive)
Contains a serine residue that is phosphorylated at the OH groups; these PO 4
groups are required for maximum catalytic activity. The PO4 groups in
phosphorylase a can be hydrolytically removed by the enzyme
phosphorulase phosphatase.
Removal
of the PO4 groups causes
phosphorylase a to dissociate into 2 half molecules, phosphorylase b, whicha
re inactive. Reactivation of the inactive phosphorylase b to form active
phosphorylase a can be brought about by the enzyme phosphorylae kinase,
which catalyses the enzymatic PO4 lation of the serine resolves at the
expense of ATP. In this way, the activity of glycogen phosphorylase
(glycogen breakdown is regulated by the action of 2 enzymes that shift the
balance between its active and inactive forms.
The 2nd string attribute of glycogen phosphorrylase and similar regulatory
enzymes modulated by covalent modification is that they can greatly
emplify a chemical signal. All enzymes can bring about amplification, i.e.
one enzyme molecule can catalyse formation of thousands of product
molecule from a given substrate in a given period of time. However, here an
enzyme acts upon another enzyme as its substrate. One molecule of
phsophorylase Kinase can convert thousands of molecules of phosphorylase
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b into the active phosphorylae a,
which in turn can catalyse the
production of thousands of molecules of G-I-P molecules from glycogen.
Phosphoglase Kinase and phosphorylase thus constitute an amplification
cascade with 2 steps. Examples of mammalian enzymes whose activity is
altered by covalent PO4 lation-de-PO4 lation.
Activity
Low
EP
EP
EP
EP
E
E
E
E
Enzyme
Acetyl CoA carboxylase
Glycogen synthase
Pyruvate dehydrogenase
HMG CoA reductase
Glycogen phospyorylase
Citrate lyase
Pyospyorylase b Knase
HMG CoA reductase Kinase
State
High
E
E
E
E
EP
EP
EP
EP
Protein Kinase
These are converte enzymes that catalyse the ATP-dep PO4 lation of serine,
threonine or tyrosine OH groups in target proteins PO4 lation introduces a
bulky group bearing 2-ve charges, causing conformational changes that alter
the target protein’s function. Unlike a phosphoryl group, no amino acid side
chain can provide 2 –ve charges. Protein Kinases differ in size, sub-unit
structure and sub-cellular location. However, they share a common catalytic
mechanism based on a conserved catalytic core/Kinase domain of
260
amino acid residues. Theya re classified as Ser/Thr and/or Tyr specific.
They also differ in terms of the target proteins that they recognize and PO 4
late target selection depends on the presence of an amino acid sequence
within the target protein that is recognized by the Kinase. For instance,
cAMP-dependent protein Kinase phosphorylated proteins having Ser or Thr
residues that occur in an Arg- (Arg or Lys) – (any amino acid) – (Ser or Thr)
sequence segiments. Tyrosine Kinases are protein Knases that PO4 lates
Tyr-residues and occur only in multicellular organisms.
They are
components of signaling pathways involved in cell-cell communication.
Classification of Protein
Protein Kinase Class
Kinases
Activators
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1. Ser/Thr Protein Kinases
A
Cyclic nucleotide-dependent
cAMP-dependent
cGMP-dependent
cAMP
cGMP
B
Ca2+ - calmodulin (CAM) dep.
Phyosphorylase Kinase
Myosin light-chainKinase (MLCK)
Phosphorylation by P.K
CA2+ - CaM
C
Protein Kinase c(PKC)
Ca2+, diacylglycerol
Mitogen-activated protein Kinases
PO4lation
D
Kinase
by
MAPK
(MAP Kinase)
E
2
3
G-protein-coupled receptors
β-Adrenergic receptor Kinase
(BARK) Rhodopsin Kinase
Ser/Thr/Tyr protein Kinases
MAP Kinase Kinase
PO4lation by Raf
Tyr protein Kinases
A
Cytosolic tyrosine Kinases
B
Receptor tyrosine Kinases (RTKs)
Plasma membrane receptors for
hormones such as epidermal growth
factor (ECF) or platelet-derived
growth factor (PDGF)
Regulation of the Activity of Protein – Kinases and Protein
Phosphatases
Targeting of protein Kinaes to particular consensus sequence elements
within proteins creates a means to regulate these Kinases by a mechanism
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referred to as Intrasteric control. Intrasteric control occurs when a
regulatory submit has a pseudosubstrate sequence that mimics the target
sequence but lacks OH-bearing side chain at the right place. For, e.g. the
cAMP-binding regulatory sub-units of protein Kinase A possess the pseudo
substrate sequence that binds to the active site of protein Kinase A catalytic
sub-units, blocking their activity. This pseudo substrate sequence in protein
Kinase A has an alanine residue where serine occurs in the target protein.
Alanine is sterically similar to serine but lacks a phosphory latable OH
group. When the regulatory subuntis of protein Kinase A bind cAMP, they
undergo a conformational change and dissociate from the catalytic sub-units
and the active site of protein Kinase A is free to bind and PO4 late its target
proteins. In other protein Kinases, the pseudosubstrate seaquence involved
in intrasteric control and the Kinase domain are part of the same polypeptide
chain. In these cases, binding of an allosteric effector (like cAMP) induces a
conformational change in the protein that releases the pseudo-substrate
sequence from the active site of the Kinase domain and the active site could
then P04late its target. Thus, dissociation of the regulatory sub-units
activates the catalytic subunits, whereas reassociation suppresses activity.
C
C
cAMP
cAMP
+ cAMP
R
R
+2
cAMP
C
cAMP
R2 C2
Inactive
Regulation of protein phosphatases also involbves P04lation and dePO4lation phosphoprotein phosphatase inhibitor. (PP1-1) is a modulator
protein that regulates the activity of phospho-protein phosphatase. When
PPI-1 is PO4lated on one of its serine residues, it binds to phosphor-protein
phosphatase, inhibiting its phosphatase activity. The result is an increased
PO4lation of the itner convertible enzyme targeted by the protein
Kinase/phosphoprotein phosphatase cycle.
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